WO2015037535A1 - Method for inducing differentiation of induced pluripotent stem cells and method for selecting induced pluripotent stem cells - Google Patents

Method for inducing differentiation of induced pluripotent stem cells and method for selecting induced pluripotent stem cells Download PDF

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WO2015037535A1
WO2015037535A1 PCT/JP2014/073511 JP2014073511W WO2015037535A1 WO 2015037535 A1 WO2015037535 A1 WO 2015037535A1 JP 2014073511 W JP2014073511 W JP 2014073511W WO 2015037535 A1 WO2015037535 A1 WO 2015037535A1
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cells
cell
pluripotent stem
induced pluripotent
derived
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French (fr)
Japanese (ja)
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一人 里村
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株式会社カネカ
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Priority to EP14844430.0A priority Critical patent/EP3045531B1/en
Priority to CN201480056130.5A priority patent/CN105849256A/en
Priority to JP2015536561A priority patent/JP6611170B2/en
Priority to US15/021,615 priority patent/US20160222347A1/en
Publication of WO2015037535A1 publication Critical patent/WO2015037535A1/en

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for inducing differentiation of induced pluripotent stem cells, a method for selecting induced pluripotent stem cells, and a kit for selecting induced pluripotent stem cells.
  • Stem cells play an important role in regenerative medicine.
  • Stem cells with universal differentiation include embryonic stem cells (ES cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), nuclear transplant ES cells, somatic cell-derived ES cells (ntES cells) and Artificial pluripotent stem cells (iPS cells) are known, and somatic stem cells, tissue stem cells, and adult stem cells are known as stem cells having pluripotent pluripotency.
  • iPS cells induced pluripotent stem cells
  • iPS cells are pluripotent and have no ethical problems associated with the destruction of embryos and eggs because they are artificially produced from somatic cells. Since there is no problem of compatibility at the time of transplantation, application to regenerative medicine is expected.
  • iPS cells induced pluripotent stem cells
  • pancreatic cells pancreatic cells, hepatocytes, cardiomyocytes, blood cells, germ cells, nerve cells, etc.
  • iPS cells induced pluripotent stem cells
  • Patent Document 1 discloses a method for inducing differentiation of a stem cell into a specific cell line, in which the stem cell is differentiated into a specific cell line in the presence of a tissue sample and / or an extracellular medium of the tissue sample. A method is described that includes culturing in vitro under inducing conditions, wherein the differentiated stem cells are of the same cell type as the tissue sample.
  • the stem cells used in Patent Document 1 are embryonic stem cells (ES cells), and there is no description regarding induced pluripotent stem cells (iPS cells).
  • Another problem is to establish a method for evaluating and managing the quality of iPS cells. That is, the present invention provides a method for efficiently inducing differentiation of iPS cells into cells having a target function, and a method for selecting iPS cells having high differentiation induction efficiency into target cells from the prepared iPS cells. It was set as a problem to be solved. Furthermore, another object of the present invention is to provide an iPS cell sorting kit for use in the above method.
  • the present inventors have conducted intensive research to solve the above-mentioned problems, and found that differentiation induction into a target cell can be achieved by culturing iPS cells on a frozen section of a tissue / organ targeted for regeneration.
  • the invention has been completed.
  • a method for inducing differentiation of an induced pluripotent stem cell comprising culturing the induced pluripotent stem cell on a structure containing cells and / or a component derived from the cell, and induction of differentiation with the cells in the structure
  • the differentiation induction method as described above, wherein the differentiated cell is the same cell type.
  • the method according to (1), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
  • the method according to (1) or (2), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
  • a method for producing a differentiation-induced cell comprising culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and induction of differentiation with the cell in the structure
  • the above production method wherein the prepared cells are of the same cell type.
  • the structure containing cells and / or components derived from cells is a sheet-like structure.
  • the method according to (6) or (7), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
  • the method according to any one of (5) to (7), wherein the cell is liver, brain or spinal cord.
  • a method for selecting induced pluripotent stem cells comprising the steps of: a cell in a structure and a cell induced to differentiate are of the same cell type.
  • the method according to (9), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
  • the method according to (9) or (10), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ section or a cell.
  • the method according to any one of (9) to (11), wherein the cells are liver, brain or spinal cord.
  • a kit for selecting induced pluripotent stem cells comprising at least a structure containing cells and / or components derived from cells.
  • the cell and / or the structure containing the component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
  • the kit according to any one of (13) to (15), wherein the cells are liver, brain or spinal cord.
  • the method of the present invention it is possible to induce differentiation of iPS cells into cells having a target function with high efficiency. Furthermore, according to the method of the present invention, it is possible to select iPS cells having high efficiency of inducing differentiation into target cells from the prepared iPS cells.
  • FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on cover glass (control), normal liver section, or hepatitis liver section.
  • FIG. 2 shows microscopic images (3rd and 9th day of culture) of iPS cells cultured on cover glass (control) or normal liver sections.
  • FIG. 3 shows microscopic images of iPS cells cultured on brain sections or spinal cord sections (3rd day and 9th day of culture).
  • FIG. 4 shows the results of examining the expression of genes related to hepatocytes (AFP, AAT, ALB) by RT-PCR.
  • FIG. 5 shows the results of examining the expression of genes related to neurons (Nestin, MBP, CNPase, and GFAP) by RT-PCR.
  • FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on cover glass (control), normal liver section, or hepatitis liver section.
  • FIG. 2 shows microscopic images (3
  • FIG. 6 shows the results of immunocytochemical analysis of AFP expression.
  • FIG. 7 shows the results of measuring the ratio of AFP positive cells in nucleated cells.
  • FIG. 8 shows the results of immunocytochemical analysis of AAT expression.
  • FIG. 9 shows the results of measuring the ratio of AAT positive cells in nucleated cells.
  • FIG. 10 shows the results of immunocytochemical analysis of GFAP expression.
  • FIG. 11 shows the results of measuring the ratio of GFAP positive cells in nucleated cells.
  • FIG. 12 shows the results of immunocytochemical analysis of CNPase expression.
  • FIG. 13 shows the results of immunocytochemical analysis of CNPase expression.
  • FIG. 14 shows the results of measuring the ratio of CNPase positive cells in nucleated cells.
  • the present invention comprises culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell.
  • the cells in the structure and the cells induced to differentiate are of the same cell type. Whether cells in the structure and differentiated cells are the same cell type can be determined by, for example, the same marker to be expressed.
  • Cell markers for hepatocytes include ⁇ -fetoprotein (AFP), ⁇ -1 antitrypsin (AAT), albumin (ALB), tyrosine aminotransferase (TAT), tryptophan 2,3 dioxygenase (TDO2), cytochrome P450, etc.
  • AFP ⁇ -fetoprotein
  • AAT ⁇ -1 antitrypsin
  • AAT albumin
  • TAT tyrosine aminotransferase
  • TDO2 tryptophan 2,3 dioxygenase
  • Neuronal markers include nestin Nestin, myelin basic protein (MBP), cyclic nucleotide phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and neurofilament (Neurofilament), but are particularly limited Not.
  • MBP myelin basic protein
  • CNPase cyclic nucleotide phosphodiesterase
  • GFAP glial fibrillary
  • osteoblast markers include, but are not limited to, alkaline phosphatase (ALP), osteopontin, and osteocalcin.
  • pancreatic cell markers include, but are not limited to, Pdx1, amylase, and carboxypeptidase.
  • chondrocyte markers include, but are not limited to, Sox9, type II collagen, and aggrecan.
  • cardiomyocyte markers include cardiac troponin I (cTnI), ⁇ -myosin heavy chain ( ⁇ -MHC), ⁇ cardiac actin ( ⁇ -cardiac actin), and homeobox protein Nkx-2.5. However, it is not particularly limited.
  • the form of the “structure containing a cell and / or a component derived from the cell” used in the present invention is not particularly limited, in view of culturing induced pluripotent stem cells (iPS cells) on the structure, a sheet It is preferable that it is a structure.
  • a culture substrate coated with a component derived from a living tissue, an organ section or a cell can be used as an example of the sheet-like structure containing a cell and / or a component derived from a cell.
  • the thickness of the sheet-like structure containing cells is not particularly limited, but is generally about 1 to 100 ⁇ m, preferably about 2 to 50 ⁇ m, more preferably It is about 2 to 20 ⁇ m.
  • the form of the “culture substrate” used in the present invention is not particularly limited, but is preferably a film, a plate or a cover glass.
  • Tissue or organ sections can be preferably collected from mammals (preferably mice or humans).
  • the type of tissue or organ is not particularly limited, and a tissue or cell containing cells of the same cell type as a differentiated cell type that is induced to differentiate from an induced pluripotent stem cell (iPS cell)
  • An organ section may be used.
  • tissues or organs include, but are not limited to, liver, brain, spinal cord, heart, respiratory organ, reproductive organ, kidney, pancreas, skin, muscle, and skeletal organ.
  • a liver slice may be used.
  • a slice containing nerve cells may be used.
  • components derived from cells include nucleic acids (DNA or RNA, particularly micro RNA) or proteins, and particularly nucleic acids (DNA or RNA, particularly micro RNA) or specifically expressed in the cells or Protein is preferred.
  • the induced pluripotent stem cell is transferred to a specific cell lineage, preferably a cell such as liver, nerve, lung, prostate, pancreas, mammary gland, kidney, intestine, skeleton, blood vessel, hematopoiesis, heart muscle, skeletal muscle, etc. Differentiation into lineages is induced.
  • a specific cell lineage preferably a cell such as liver, nerve, lung, prostate, pancreas, mammary gland, kidney, intestine, skeleton, blood vessel, hematopoiesis, heart muscle, skeletal muscle, etc. Differentiation into lineages is induced.
  • artificial pluripotent stem cells are established by introducing 4 genes of OCT3 / 4, SOX2, NANOG, and LIN28 into human fibroblasts (Yu J., Thomson JA.et al., Science). (2007) 318: 1917-1920.).
  • an artificial pluripotent stem cell is established by introducing 6 genes of OCT3 / 4, SOX2, KLF4, C-MYC, hTERT, SV40 large T into skin cells (Park IH, Daley GQ.et al., Nature (2007) 451: 141-146), establishing induced pluripotent stem cells by introducing Oct3 / 4, Sox2, Klf4, c-Myc, etc. into undifferentiated stem cells present in postnatal tissues rather than somatic cells (Japanese Patent Laid-Open No. 2008-307007) has also been reported.
  • an induced pluripotent stem cell is a cell having multipotency and self-proliferation ability induced by reprogramming somatic cells or undifferentiated stem cells.
  • the origin of the somatic cell is not limited, and may be derived from an embryo, fetus, or adult.
  • the animal species from which the somatic cell is derived is not particularly limited, but is preferably a mammal, more preferably a human or a mouse.
  • somatic cells include, but are not limited to, fibroblasts, epithelial cells, hepatocytes, blood cells and the like.
  • the method for producing induced pluripotent stem cells used in the present invention is not particularly limited, and the introduction factor and the introduction method are not particularly limited.
  • the feeder cells are detached and iPS cells are collected, and an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum) Suspend in Next, a culture cover glass on which a structure containing cells (preferably, a section of a living tissue or an organ) is placed may be placed, and an iPS cell suspension may be seeded therein.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • the conditions for culturing induced pluripotent stem cells on a structure containing cells and / or components derived from cells are not particularly limited as long as differentiation of induced pluripotent stem cells can be induced.
  • the medium for example, Dulbecco's modified Eagle medium (DMEM) containing fetal bovine serum, or a medium for primate ES cells (Reprocell Co., Ltd.) can be used.
  • DMEM Dulbecco's modified Eagle medium
  • Various growth factors for inducing differentiation of induced pluripotent stem cells, cytokines or differentiation inducing factors may be added and cultured.
  • by culturing on a structure containing cells. Differentiation induction can be achieved without adding the above-mentioned growth factors and differentiation induction factors.
  • Examples of various growth factors, cytokines or differentiation inducing factors for inducing differentiation of induced pluripotent stem cells include actin bin, bFGF, noggin, nicotinamide, retinoic acid, EGF, glucocorticoid, etc. There is no particular limitation.
  • induced pluripotent stem cells are induced to differentiate by culturing induced pluripotent stem cells on a structure containing cells and / or components derived from the cells.
  • induced pluripotent stem cells with high differentiation induction efficiency can be selected.
  • induced pluripotent stem cells are currently produced by various methods.
  • the created induced pluripotent stem cells are expected to be induced to differentiate into desired cells and applied to regenerative medicine.
  • the created artificial pluripotent stem cells usually contain a mixture of cells having high efficiency of inducing differentiation into desired cells and cells having low efficiency of inducing differentiation into desired cells. . It has been desired to select cells with high efficiency of inducing differentiation into desired cells from the prepared induced pluripotent stem cells.
  • an induced pluripotent stem cell is induced to differentiate by culturing the induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and the efficiency of inducing differentiation into a desired cell is improved.
  • Highly induced pluripotent stem cells can be selected.
  • the induced pluripotent stem cells selected in this way and having high differentiation-inducing efficiency can be stored as a stock, and can be differentiated and used when necessary in the field of regenerative medicine.
  • a structure containing cells and / or components derived from cells such as a section of a living tissue or an organ, can be provided as a kit for selecting induced pluripotent stem cells.
  • the kit suitably includes a medium for culturing induced pluripotent stem cells, a cover glass for installing structures containing cells, and the like. May be.
  • iPS cell culture and differentiation induction After culturing iPS cells according to a conventional method, the feeder cells are detached using an iPS / ES cell detachment solution (CTK solution, Reprocell Co., Ltd.), washed with PBS, and then iPS cells. Were collected and suspended in Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM, SIGMA) containing 5% fetal bovine serum (hereinafter abbreviated as FBS, Biological Industries). A circular culture cover glass on which the aforementioned frozen section was placed was placed on a 35 mm diameter dish (non-coated type, Matsunami glass), and a suspension of iPS cells was seeded thereon.
  • CTK solution iPS / ES cell detachment solution
  • FBS fetal bovine serum
  • FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on a cover glass (control), a normal liver section, or a hepatitis liver section. It was observed that the colonies of iPS cells seeded in the control, normal liver, and hepatitis liver spread and the cells spread. In addition, changes in cell morphology were also observed. In the control, iPS cells that had expanded on the 8th day compared to the 2nd day of culture exhibited various forms as indicated by arrows, and no uniformity was observed. On the other hand, in the normal liver and hepatitis liver, the expanded cells showed a relatively large and polygonal shape as indicated by arrows.
  • FIG. 2 a microscopic image (3rd and 9th culture days) of iPS cells cultured on a cover glass (control) or a normal liver slice is shown in FIG. 2, and the iPS cells cultured on a brain slice or spinal cord slice are shown.
  • a microscopic image (3rd day and 9th day of culture) is shown in FIG.
  • RNA expression of various differentiation markers of neurons and hepatocytes was collected from iPS cells cultured on frozen sections of liver, brain, and spinal cord using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA).
  • RNA obtained 1 ⁇ g was treated with 100 units / ml deoxyribonuclease I (hereinafter abbreviated as DNase I) at room temperature, and then 1 ml of 25 mM EDTA was added and treated at 65 ° C. for 5 minutes to lose DNase I. I made it live.
  • a random hexamer primer (Invitrogen) was added, and a reverse transcription reaction was performed at 50 ° C. for 50 minutes using the reverse transcriptase SUPERSCRIPT III Preamplification System (Invitrogen). Further, it was treated with ribonuclease H at 37 ° C. for 20 minutes to obtain cDNA.
  • PCR was performed using primers specific to each human gene and PCR MasterMix Kit (Thermo, Rockford, USA) to examine mRNA expression of each gene.
  • the examined differentiation markers, primers and PCR conditions are shown in Table 1.
  • PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and then visualized using a UV imaging device FAS-III (Toyobo, Osaka).
  • hepatocyte-related genes AFP, AAT, ALB
  • the expression of AFP was observed in the control group, normal liver group, and hepatitis liver group, but the expression was stronger in the hepatitis liver group than in the control group, and the expression was further enhanced in the normal liver group.
  • AAT expression was also observed in the control group, normal liver group, and hepatitis liver group, but the expression was strongly observed in the normal liver group as compared with the control group.
  • the expression of ALB was not observed in the control group, whereas the expression was confirmed in the normal liver group and hepatitis liver group, and the expression was stronger in the normal liver group than in the hepatitis liver group. It was.
  • GAPDH Glyceraldehyde-3-phosphate dehydrogenase AFP: ⁇ -fetoprotein AAT: ⁇ 1-antitrypsin ALB: Albumin MBP: Myelin basic protein CNPase: 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase GFAP: Glial fibrous acidic protein NES: Nestin
  • FITC-conjugated anti-rabbit IgG or FITC-conjugated anti-goat IgG was reacted as a secondary antibody for 1 hour at room temperature.
  • nuclei were stained with DAPI and observed with a fluorescence microscope (FIGS. 6 and 8).
  • FIG. 6 almost no AFP positive cells were observed in the control group, but many AFP positive cells were observed in the normal liver group and the hepatitis liver group.
  • FIG. 8 as in the case of AFP, more AAT positive cells were observed in the normal liver group and the hepatitis liver group than in the control group.
  • the differentiation induction efficiency was examined by measuring the number of iPS cells expressing ⁇ -fetoprotein and ⁇ 1-antitrypsin and calculating the proportion of nucleated cells (FIGS. 7 and 9). As shown in FIG. 7, the ratio of AFP positive cells was significantly higher in the normal liver group and the group on the first day of hepatitis liver than in the control group. In addition, as shown in FIG. 9, the proportion of AAT positive cells was significantly higher in the normal liver group and the hepatitis liver 1, 2 and 5 day groups than in the control group.
  • IPS cells cultured on various frozen sections were fixed with 2% paraformaldehyde for 1 hour, washed twice with PBS, and then diluted with 0.1% BSA-containing PBS to 1% TRITON X-100 (ICN Biomedical). For 15 minutes. After washing twice with PBS, blocking was performed with 10% normal goat serum for 1 hour. After washing twice with PBS, the mixture was reacted overnight at 4 ° C. with a primary antibody diluted with PBS containing 1% BSA.
  • Rabbit anti-Glial Bibrillary Acidic Protein antibody SIGMA, 80-fold dilution
  • Rabbit anti-CNPase antibody Abcam, 100-fold dilution
  • a secondary antibody Goat anti-rabbit IgG FITC (Wako, diluted 40-fold) was used for GFAP
  • Goat anti-rabbit IgG Alexa Invitergen, diluted 100-fold was used for CNPase.
  • FIG. 10 shows the results of observation of protein expression with a fluorescence microscope using an anti-human GFAP antibody as a primary antibody.
  • the number of iPS cells expressing GFAP was counted, and the proportion of nucleated cells was calculated (FIG. 11), and the differentiation induction efficiency was examined.
  • the results of observation of protein expression with a fluorescence microscope using an anti-CNPase antibody as the primary antibody are shown in FIGS.
  • the differentiation induction efficiency was examined by measuring the number of iPS cells expressing CNPase and calculating the proportion of nucleated cells (FIG. 14).

Abstract

The present invention addresses the problem of providing a method for highly efficiently inducing differentiation of induced pluripotent stem cells (iPS cells) into cells having a desired function, and a method for selecting, from among the iPS cells prepared above, iPS cells that can be highly efficiently induced to undergo differentiation into the target cells. The method for inducing differentiation of iPS cells, said method comprising culturing iPS cells on a structure that contains a cell and/or a component derived from a cell, wherein the cell in the structure is of the same cell type as the cells having been induced to undergo differentiation.

Description

人工多能性幹細胞の分化誘導方法及び選別方法Method for inducing differentiation and selection of induced pluripotent stem cells
　本発明は、人工多能性幹細胞の分化誘導方法、人工多能性幹細胞の選別方法、並びに人工多能性幹細胞の選別キットに関する。 The present invention relates to a method for inducing differentiation of induced pluripotent stem cells, a method for selecting induced pluripotent stem cells, and a kit for selecting induced pluripotent stem cells.
　幹細胞は再生医療において重要な役割を担う。分化万能性を有する幹細胞としては、胚性幹細胞（ES細胞）、胚性腫瘍細胞（EC細胞）、胚性生殖幹細胞（EG細胞）、核移植ES細胞、体細胞由来ES細胞（ntES細胞）及び人工多能性幹細胞（iPS細胞）が知られており、 分化多能性を有する幹細胞としては、体性幹細胞、組織幹細胞及び成体幹細胞が知られている。上記の中でも、人工多能性幹細胞（iPS細胞）は、分化万能性を有し、体細胞から人工的に作製される細胞であることから胚や卵子の破壊に伴う倫理上の問題がなく、移植の際の適合性の問題もないことから、再生医療への応用が期待されている。 Stem cells play an important role in regenerative medicine. Stem cells with universal differentiation include embryonic stem cells (ES cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), nuclear transplant ES cells, somatic cell-derived ES cells (ntES cells) and Artificial pluripotent stem cells (iPS cells) are known, and somatic stem cells, tissue stem cells, and adult stem cells are known as stem cells having pluripotent pluripotency. Among the above, induced pluripotent stem cells (iPS cells) are pluripotent and have no ethical problems associated with the destruction of embryos and eggs because they are artificially produced from somatic cells. Since there is no problem of compatibility at the time of transplantation, application to regenerative medicine is expected.
　人工多能性幹細胞（iPS細胞）を所望の細胞に分化誘導する方法は、各種方法が報告されており、例えば、膵細胞、肝細胞、心筋細胞、血液細胞、生殖細胞、神経細胞などへの分化誘導が報告されている。しかし、人工多能性幹細胞（iPS細胞）を用いた再生医療を実用化するにあたっては、高効率に目的の機能を有する細胞に分化誘導する方法を確立することが必要である。 Various methods have been reported for inducing differentiation of induced pluripotent stem cells (iPS cells) into desired cells. For example, pancreatic cells, hepatocytes, cardiomyocytes, blood cells, germ cells, nerve cells, etc. Differentiation induction has been reported. However, in order to put regenerative medicine using artificial pluripotent stem cells (iPS cells) into practical use, it is necessary to establish a method for inducing differentiation into cells having a target function with high efficiency.
　特許文献１には、幹細胞の特定細胞系統への分化を誘発する方法であって、幹細胞を、組織サンプル及び／又は組織サンプルの細胞外培地の存在下、該幹細胞の特定細胞系統への分化を誘発する条件下で、in vitro で培養することを含み、その際、該分化した幹細胞が該組織サンプルと同じ細胞型である方法が記載されている。特許文献１で使用されている幹細胞は胚性幹細胞（ＥＳ細胞）であり、人工多能性幹細胞（iPS細胞）に関する記載はない。 Patent Document 1 discloses a method for inducing differentiation of a stem cell into a specific cell line, in which the stem cell is differentiated into a specific cell line in the presence of a tissue sample and / or an extracellular medium of the tissue sample. A method is described that includes culturing in vitro under inducing conditions, wherein the differentiated stem cells are of the same cell type as the tissue sample. The stem cells used in Patent Document 1 are embryonic stem cells (ES cells), and there is no description regarding induced pluripotent stem cells (iPS cells).
特表２００５－５２０５１６号公報JP 2005-520516 A
　人工多能性幹細胞（iPS細胞）を用いた再生医療を実用化する際における課題として、未分化状態のiPS細胞を、目的の機能を有する細胞に高効率に分化誘導する技術を確立することが挙げられる。また、別の課題としては、iPS細胞の品質の評価・管理する方法を確立することが挙げられる。即ち、本発明は、iPS細胞を目的の機能を有する細胞に高効率に分化誘導する方法、並びに作製したiPS細胞の中から目的とする細胞への分化誘導効率が高いiPS細胞を選別する方法を提供することを解決すべき課題とした。更に本発明は、上記方法で使用するためのiPS細胞選別キットを提供することを解決すべき課題とした。 Establishing a technology that induces differentiation of undifferentiated iPS cells into cells with the desired functions with high efficiency as a challenge in the practical application of regenerative medicine using artificial pluripotent stem cells (iPS cells) Can be mentioned. Another problem is to establish a method for evaluating and managing the quality of iPS cells. That is, the present invention provides a method for efficiently inducing differentiation of iPS cells into cells having a target function, and a method for selecting iPS cells having high differentiation induction efficiency into target cells from the prepared iPS cells. It was set as a problem to be solved. Furthermore, another object of the present invention is to provide an iPS cell sorting kit for use in the above method.
　本発明者らは、前記課題を解決すべく鋭意研究を行い、再生を目指す組織・臓器の凍結切片上でiPS細胞を培養することにより目的とする細胞への分化誘導を達成できることを見出し、本発明を完成するに至った。 The present inventors have conducted intensive research to solve the above-mentioned problems, and found that differentiation induction into a target cell can be achieved by culturing iPS cells on a frozen section of a tissue / organ targeted for regeneration. The invention has been completed.
　すわなち、本発明によれば以下の発明が提供される。
（１）　細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の分化誘導方法。
（２）　細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、（１）に記載の方法。
（３）　細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、（１）又は（２）に記載の方法。
（４）　細胞が、肝臓、脳又は脊髄である、（１）から（３）の何れかに記載の方法。 That is, according to the present invention, the following inventions are provided.
(1) A method for inducing differentiation of an induced pluripotent stem cell, comprising culturing the induced pluripotent stem cell on a structure containing cells and / or a component derived from the cell, and induction of differentiation with the cells in the structure The differentiation induction method as described above, wherein the differentiated cell is the same cell type.
(2) The method according to (1), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
(3) The method according to (1) or (2), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
(4) The method according to any one of (1) to (3), wherein the cells are liver, brain or spinal cord.
（５）　細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む、分化誘導された細胞の製造方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の製造方法。
（６）　細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、（５）に記載の方法。
（７）　細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、（６）又は（７）に記載の方法。
（８）　細胞が、肝臓、脳又は脊髄である、（５）から（７）の何れかに記載の方法。 (5) A method for producing a differentiation-induced cell, comprising culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and induction of differentiation with the cell in the structure The above production method, wherein the prepared cells are of the same cell type.
(6) The method according to (5), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
(7) The method according to (6) or (7), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
(8) The method according to any one of (5) to (7), wherein the cell is liver, brain or spinal cord.
（９）　細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、分化誘導効率が高い人工多能性幹細胞を選別することを含む人工多能性幹細胞の選別方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の選別方法。
（１０）　細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、（９）に記載の方法。
（１１）　細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、（９）又は（１０）に記載の方法。
（１２）　細胞が、肝臓、脳又は脊髄である、（９）から（１１）の何れかに記載の方法。 (9) Inducing induced pluripotent stem cells by culturing induced pluripotent stem cells on structures containing cells and / or cell-derived components, and selecting induced pluripotent stem cells with high differentiation induction efficiency A method for selecting induced pluripotent stem cells, comprising the steps of: a cell in a structure and a cell induced to differentiate are of the same cell type.
(10) The method according to (9), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
(11) The method according to (9) or (10), wherein the structure containing a cell and / or a component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ section or a cell.
(12) The method according to any one of (9) to (11), wherein the cells are liver, brain or spinal cord.
（１３）　細胞及び／又は細胞に由来する成分を含む構造物を少なくとも含む、人工多能性幹細胞の選別のためのキット。
（１４）　細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、（１３）に記載のキット。
（１５）　細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、（１３）又は（１４）に記載のキット。
（１６）　細胞が、肝臓、脳又は脊髄である、（１３）から（１５）の何れかに記載のキット。 (13) A kit for selecting induced pluripotent stem cells, comprising at least a structure containing cells and / or components derived from cells.
(14) The kit according to (13), wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
(15) The kit according to (13) or (14), wherein the cell and / or the structure containing the component derived from the cell is a culture substrate coated with a tissue derived from a living tissue, an organ slice or a cell.
(16) The kit according to any one of (13) to (15), wherein the cells are liver, brain or spinal cord.
　本発明の方法によれば、iPS細胞を目的の機能を有する細胞に高効率に分化誘導することが可能である。さらに本発明の方法によれば、作製したiPS細胞の中から目的とする細胞への分化誘導効率が高いiPS細胞を選別することが可能である。 According to the method of the present invention, it is possible to induce differentiation of iPS cells into cells having a target function with high efficiency. Furthermore, according to the method of the present invention, it is possible to select iPS cells having high efficiency of inducing differentiation into target cells from the prepared iPS cells.
図１は、カバーグラス上（対照）、正常肝臓切片上、又は肝炎肝臓切片上で培養したiPS細胞の顕微鏡像（培養２日目と培養８日目）を示す。FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on cover glass (control), normal liver section, or hepatitis liver section. 図２は、カバーグラス上（対照）又は正常肝臓切片上で培養したiPS細胞の顕微鏡像（培養３日目と９日目）を示す。FIG. 2 shows microscopic images (3rd and 9th day of culture) of iPS cells cultured on cover glass (control) or normal liver sections. 図３は、脳切片上又は脊髄切片上で培養したiPS細胞の顕微鏡像（培養３日目と９日目）を示す。FIG. 3 shows microscopic images of iPS cells cultured on brain sections or spinal cord sections (3rd day and 9th day of culture). 図４は、肝細胞の関連遺伝子（ＡＦＰ、ＡＡＴ、ＡＬＢ）の発現をＲＴ－ＰＣＲにより調べた結果を図４に示す。FIG. 4 shows the results of examining the expression of genes related to hepatocytes (AFP, AAT, ALB) by RT-PCR. 図５は、神経細胞の関連遺伝子（Nestin、ＭＢＰ、CNPase、及びGFAP）の発現をＲＴ－ＰＣＲにより調べた結果を示す。FIG. 5 shows the results of examining the expression of genes related to neurons (Nestin, MBP, CNPase, and GFAP) by RT-PCR. 図６は、ＡＦＰの発現を免疫細胞化学的に分析した結果を示す。FIG. 6 shows the results of immunocytochemical analysis of AFP expression. 図７は、有核細胞におけるＡＦＰ陽性細胞の割合を計測した結果を示す。FIG. 7 shows the results of measuring the ratio of AFP positive cells in nucleated cells. 図８は、ＡＡＴの発現を免疫細胞化学的に分析した結果を示す。FIG. 8 shows the results of immunocytochemical analysis of AAT expression. 図９は、有核細胞におけるＡＡＴ陽性細胞の割合を計測した結果を示す。FIG. 9 shows the results of measuring the ratio of AAT positive cells in nucleated cells. 図１０は、ＧＦＡＰの発現を免疫細胞化学的に分析した結果を示す。FIG. 10 shows the results of immunocytochemical analysis of GFAP expression. 図１１は、有核細胞におけるＧＦＡＰ陽性細胞の割合を計測した結果を示す。FIG. 11 shows the results of measuring the ratio of GFAP positive cells in nucleated cells. 図１２は、ＣＮＰａｓｅの発現を免疫細胞化学的に分析した結果を示す。FIG. 12 shows the results of immunocytochemical analysis of CNPase expression. 図１３は、ＣＮＰａｓｅの発現を免疫細胞化学的に分析した結果を示す。FIG. 13 shows the results of immunocytochemical analysis of CNPase expression. 図１４は、有核細胞におけるＣＮＰａｓｅ陽性細胞の割合を計測した結果を示す。FIG. 14 shows the results of measuring the ratio of CNPase positive cells in nucleated cells.
　以下、本発明について更に詳細に説明する。
（１）人工多能性幹細胞の分化誘導方、並びに分化誘導された細胞の製造方法
　本発明は、細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法又は分化誘導された細胞の製造方法に関し、特に、構造物中の細胞と分化誘導された細胞とが同じ細胞型である。なお、構造物中の細胞と分化誘導された細胞とが同じ細胞型であるかどうかは、例えば、発現するマーカーが同じであることにより判断することができる。 Hereinafter, the present invention will be described in more detail.
(1) Differentiation induction method of induced pluripotent stem cell and method for producing differentiation induced cell The present invention comprises culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell. In particular, the cells in the structure and the cells induced to differentiate are of the same cell type. Whether cells in the structure and differentiated cells are the same cell type can be determined by, for example, the same marker to be expressed.
　肝細胞の細胞マーカーとしては、α－フェトプロテイン（ＡＦＰ）、α－１アンチトリプシン（ＡＡＴ）、アルブミン（ＡＬＢ）、チロシンアミノトランスフェラーゼ(TAT)、トリプトファン2,3ジオキシゲナーゼ(TDO2)、及びシトクロムP450などが挙げられるが、特に限定されない。
　神経細胞のマーカーとしては、ネスチンNestin）、ミエリン塩基性蛋白（ＭＢＰ）、環状ヌクレオチドホスホジエステラーゼ（CNPase）、グリア細胞線維性酸性タンパク質（GFAP）、及びニューロフィラメント（Neurofilament）などが挙げられるが、特に限定されない。 Cell markers for hepatocytes include α-fetoprotein (AFP), α-1 antitrypsin (AAT), albumin (ALB), tyrosine aminotransferase (TAT), tryptophan 2,3 dioxygenase (TDO2), cytochrome P450, etc. However, it is not particularly limited.
Neuronal markers include nestin Nestin, myelin basic protein (MBP), cyclic nucleotide phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and neurofilament (Neurofilament), but are particularly limited Not.
　骨芽細胞のマーカーとしては、アルカリフォスファターゼ（ＡＬＰ）、オステオポンチン、及びオステオカルシンなどが挙げられるが、特に限定されない。
　膵臓細胞のマーカーとしては、Pdx1、アミラーゼ、及びカルボキシペプチターゼなどが挙げられるが、特に限定されない。 Examples of osteoblast markers include, but are not limited to, alkaline phosphatase (ALP), osteopontin, and osteocalcin.
Examples of pancreatic cell markers include, but are not limited to, Pdx1, amylase, and carboxypeptidase.
　軟骨細胞のマーカーとしては、Sox9、II型コラーゲン、及びアグリカンなどが挙げられるが、特に限定されない。
　心筋細胞のマーカーとしては、心筋型トロポニンI（cardiac troponin I：cTnI）、α－ミオシン重鎖（α－ＭＨＣ）、α心筋アクチン（α－cardiac actin）、及びホメオボックスプロテインNkx-2.5などが挙げられるが、特に限定されない。 Examples of chondrocyte markers include, but are not limited to, Sox9, type II collagen, and aggrecan.
Examples of cardiomyocyte markers include cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), α cardiac actin (α-cardiac actin), and homeobox protein Nkx-2.5. However, it is not particularly limited.
　本発明で用いる「細胞及び／又は細胞に由来する成分を含む構造物」の形態は特に限定されないが、当該構造物上で人工多能性幹細胞（iPS細胞）を培養することを鑑みると、シート状構造物であることが好ましい。細胞及び／又は細胞に由来する成分を含むシート状構造物の一例としては、生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材を使用することができる。細胞を含むシート状構造物（好ましくは、生体組織又は臓器の切片）の厚さは特に限定されないが、一般的には１～１００μｍ程度であり、好ましくは２～５０μｍ程度であり、より好ましくは２～２０μｍ程度である。
　細胞に由来する成分を含む構造物は、細胞に由来する成分と相互作用を有する核酸、マイクロＲＮＡ、抗体または化合物を用いて細胞に由来する成分を精製し、精製した細胞に由来する成分を培養基材上に付着させ、酵素や界面活性剤等を用いてコーティングさせることで得ることができる。また、コーティングした培養基材をさらに乾燥させることで、長期に保存可能な培養基材を提供できる。
　本発明で用いる「培養基材」の形態は特に限定されないが、フィルム、プレートまたはカバーガラスであることが好ましい。 Although the form of the “structure containing a cell and / or a component derived from the cell” used in the present invention is not particularly limited, in view of culturing induced pluripotent stem cells (iPS cells) on the structure, a sheet It is preferable that it is a structure. As an example of the sheet-like structure containing a cell and / or a component derived from a cell, a culture substrate coated with a component derived from a living tissue, an organ section or a cell can be used. The thickness of the sheet-like structure containing cells (preferably, a section of a living tissue or organ) is not particularly limited, but is generally about 1 to 100 μm, preferably about 2 to 50 μm, more preferably It is about 2 to 20 μm.
For structures containing cell-derived components, purify cell-derived components using nucleic acids, microRNAs, antibodies, or compounds that interact with cell-derived components, and culture the purified cell-derived components. It can be obtained by depositing on a substrate and coating with an enzyme, a surfactant or the like. Further, by further drying the coated culture substrate, a culture substrate that can be stored for a long period of time can be provided.
The form of the “culture substrate” used in the present invention is not particularly limited, but is preferably a film, a plate or a cover glass.
　組織又は臓器の切片は、好ましくは哺乳動物（好ましくは、マウス又はヒトなど）から採取することができる。生体組織又は臓器の切片を使用する場合、組織又は臓器の種類は特に限定されず、人工多能性幹細胞（iPS細胞）から分化誘導する分化細胞の細胞型と同じ細胞型の細胞を含む組織又は臓器の切片を使用すればよい。組織又は臓器の例としては、肝臓、脳、脊髄、心臓、呼吸器、生殖器官、腎臓、膵臓、皮膚、筋肉及び骨格器官などを挙げることができるが、特に限定されない。例えば、iPS細胞から肝細胞に分化誘導することを意図する場合には、肝臓切片を使用すればよく、iPS細胞から神経細胞に分化誘導することを意図する場合には、神経細胞を含む切片（例えば、脳切片又は脊髄切片など）を使用すればよい。 Tissue or organ sections can be preferably collected from mammals (preferably mice or humans). When using a section of a biological tissue or organ, the type of tissue or organ is not particularly limited, and a tissue or cell containing cells of the same cell type as a differentiated cell type that is induced to differentiate from an induced pluripotent stem cell (iPS cell) An organ section may be used. Examples of tissues or organs include, but are not limited to, liver, brain, spinal cord, heart, respiratory organ, reproductive organ, kidney, pancreas, skin, muscle, and skeletal organ. For example, when it is intended to induce differentiation from iPS cells to hepatocytes, a liver slice may be used. When it is intended to induce differentiation from iPS cells to nerve cells, a slice containing nerve cells ( For example, a brain slice or spinal cord slice) may be used.
　細胞に由来する成分としては、核酸（ＤＮＡ又はＲＮＡ、特にマイクロＲＮＡなど）又はタンパク質などを挙げられ、特に上記細胞に特異的に発現されているような核酸（ＤＮＡ又はＲＮＡ、特にマイクロＲＮＡ）又はタンパク質が好ましい。 Examples of components derived from cells include nucleic acids (DNA or RNA, particularly micro RNA) or proteins, and particularly nucleic acids (DNA or RNA, particularly micro RNA) or specifically expressed in the cells or Protein is preferred.
　本発明の方法では、人工多能性幹細胞は、特定細胞系統へ、好ましくは、肝臓、神経、肺、前立腺、膵臓、乳腺、腎臓、腸、骨格、血管、造血、心筋、骨格筋などの細胞系統へ分化誘導される。 In the method of the present invention, the induced pluripotent stem cell is transferred to a specific cell lineage, preferably a cell such as liver, nerve, lung, prostate, pancreas, mammary gland, kidney, intestine, skeleton, blood vessel, hematopoiesis, heart muscle, skeletal muscle, etc. Differentiation into lineages is induced.
　人工多能性幹細胞は、マウス繊維芽細胞にＯｃｔ３／４、Ｓｏｘ２、Ｋｌｆ４、ｃ－Ｍｙｃの４因子を導入することにより初めて樹立されｉＰＳ細胞と命名された（Takahashi K,Yamanaka S.,Cell,(2006)126:663-676）。その後、同様の４因子を用いてヒトｉＰＳも樹立されている（Takahashi K,Yamanaka S.,et al.Cell,(2007)131:861-872.）。さらにｃ－Ｍｙｃを含まない３因子を用いる方法（Nakagawa M,Yamanaka S.,et al.Nature Biotechnology,(2008)26,101-106）も報告されている。また、ＯＣＴ３／４，ＳＯＸ２，ＮＡＮＯＧ，ＬＩＮ２８の４遺伝子をヒト繊維芽細胞に導入して人工多能性幹細胞を樹立することも報告されている（Yu J.,Thomson JA.et al.,Science(2007)318:1917-1920.）。また、皮膚細胞にＯＣＴ３／４，ＳＯＸ２，ＫＬＦ４，Ｃ－ＭＹＣ，ｈＴＥＲＴ，ＳＶ４０ ｌａｒｇｅ Ｔの６遺伝子を導入して人工多能性幹細胞を樹立すること（Park IH,Daley GQ.et al.,Nature(2007)451:141-146）、体細胞ではなく出生後の組織に存在する未分化幹細胞にＯｃｔ３／４、Ｓｏｘ２、Ｋｌｆ４及びｃ－Ｍｙｃ等を導入することで人工多能性幹細胞を樹立すること（特開２００８－３０７００７）も報告されている。 Artificial pluripotent stem cells were established for the first time by introducing four factors Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts and named iPS cells (TakahashiYK, Yamanaka S., Cell, (2006) 126: 663-676). Subsequently, human iPS was also established using the same four factors (Takahashi K, Yamanaka S., et al.Cell, (2007) 131: 861-872.). Furthermore, a method using three factors not containing c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106) has also been reported. It has also been reported that artificial pluripotent stem cells are established by introducing 4 genes of OCT3 / 4, SOX2, NANOG, and LIN28 into human fibroblasts (Yu J., Thomson JA.et al., Science). (2007) 318: 1917-1920.). In addition, an artificial pluripotent stem cell is established by introducing 6 genes of OCT3 / 4, SOX2, KLF4, C-MYC, hTERT, SV40 large T into skin cells (Park IH, Daley GQ.et al., Nature (2007) 451: 141-146), establishing induced pluripotent stem cells by introducing Oct3 / 4, Sox2, Klf4, c-Myc, etc. into undifferentiated stem cells present in postnatal tissues rather than somatic cells (Japanese Patent Laid-Open No. 2008-307007) has also been reported.
　上記の通り、人工多能性幹細胞（iPS細胞）とは、体細胞又は未分化な幹細胞を初期化することにより誘導された多分化能及び自己増殖能を有する細胞である。体細胞の由来は限定されず、胚由来、胎児由来、又は成体由来の何れでもよい。体細胞が由来する動物種も特に限定されないが、哺乳動物が好ましく、ヒト又はマウスなどがさらに好ましい。体細胞としては、例えば、繊維芽細胞、上皮細胞、肝細胞、血液細胞などが挙げられるが、特に限定されない。本発明で使用する人工多能性幹細胞の製造方法は特に限定されず、導入因子や導入方法等も特に限定されない。人工多能性幹細胞に関する公知の文献としては、上記以外にも、特開２００８－２８３９７２、ＵＳ２００８－２３３６６１０、ＵＳ２００９－０４７２６３、ＷＯ２００７／０６９６６６、ＷＯ２００８／１１８２２０、ＷＯ２００８／１２４１３３、ＷＯ２００８／１５１０５８、ＷＯ２００９／０５７８３１、ＷＯ２００９／００６９９７、ＷＯ２００９／００７８５２等を挙げることができる。 As described above, an induced pluripotent stem cell (iPS cell) is a cell having multipotency and self-proliferation ability induced by reprogramming somatic cells or undifferentiated stem cells. The origin of the somatic cell is not limited, and may be derived from an embryo, fetus, or adult. The animal species from which the somatic cell is derived is not particularly limited, but is preferably a mammal, more preferably a human or a mouse. Examples of somatic cells include, but are not limited to, fibroblasts, epithelial cells, hepatocytes, blood cells and the like. The method for producing induced pluripotent stem cells used in the present invention is not particularly limited, and the introduction factor and the introduction method are not particularly limited. In addition to the above, publicly known documents related to induced pluripotent stem cells include JP 2008-283972, US 2008-2336610, US 2009-047263, WO 2007/069666, WO 2008/118220, WO 2008/124133, WO 2008/151088, WO 2009/057831. , WO2009 / 006997, WO2009 / 007852 and the like.
　本発明においては、常法に従いフィーダー細胞上においてiPS細胞を培養後に、フィーダー細胞を剥離してiPS細胞を回収し、適当な培地（例えば、ウシ胎児血清を含むダルベッコ改変イーグル培地（DMEM）など）に懸濁する。次いで、細胞を含む構造物（好ましくは、生体組織又は臓器の切片）を載せたカルチャーカバーグラスを設置し、ここにiPS細胞の懸濁液を播種すればよい。播種後静置することで、浮遊しているiPS細胞を含む構造物へ付着させ、その後、適当な培地（5%FBSを含むDMEMなど）を加え、常法に従って培養を行えばよい。 In the present invention, after culturing iPS cells on feeder cells according to a conventional method, the feeder cells are detached and iPS cells are collected, and an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum) Suspend in Next, a culture cover glass on which a structure containing cells (preferably, a section of a living tissue or an organ) is placed may be placed, and an iPS cell suspension may be seeded therein. By allowing to stand after seeding, the cells are allowed to adhere to a structure containing floating iPS cells, and then an appropriate medium (such as DMEM containing 5% FBS) is added, followed by culturing according to a conventional method.
　細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養する際の条件は、人工多能性幹細胞の分化を誘導できる限り、特に限定はない。培地としては、例えば、ウシ胎児血清を含むダルベッコ改変イーグル培地（DMEM）、又は霊長類ES細胞用培地（株式会社リプロセル）などを使用することができる。人工多能性幹細胞の分化を誘導するための各種の増殖因子、サイトカイン又は分化誘導因子などを添加して培養してもよいが、本発明においては細胞を含む構造物上で培養を行うことにより、上記した増殖因子や分化誘導因子を添加しなくても分化誘導を達成することができる。人工多能性幹細胞の分化を誘導するための各種の増殖因子、サイトカイン又は分化誘導因子の例としては、アクチンビン、bFGF、ノギン、ニコチンアミド、レチノイン酸、EGF、グルココルチコイドなどを挙げることができるが特に限定はされない。 The conditions for culturing induced pluripotent stem cells on a structure containing cells and / or components derived from cells are not particularly limited as long as differentiation of induced pluripotent stem cells can be induced. As the medium, for example, Dulbecco's modified Eagle medium (DMEM) containing fetal bovine serum, or a medium for primate ES cells (Reprocell Co., Ltd.) can be used. Various growth factors for inducing differentiation of induced pluripotent stem cells, cytokines or differentiation inducing factors may be added and cultured. In the present invention, by culturing on a structure containing cells. Differentiation induction can be achieved without adding the above-mentioned growth factors and differentiation induction factors. Examples of various growth factors, cytokines or differentiation inducing factors for inducing differentiation of induced pluripotent stem cells include actin bin, bFGF, noggin, nicotinamide, retinoic acid, EGF, glucocorticoid, etc. There is no particular limitation.
（２）人工多能性幹細胞の選別方法
　本発明によれば、細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、分化誘導効率が高い人工多能性幹細胞を選別することができる。上記した通り、人工多能性幹細胞は現在、様々な方法で作成されている。作成された人工多能性幹細胞は、所望の細胞へと分化誘導され、再生医療などに適用されることが期待されている。しかしながら、作成された人工多能性幹細胞の中には、所望の細胞への分化誘導効率が高い細胞と、所望の細胞への分化誘導効率が低い細胞とが混在しているのが普通である。作成した人工多能性幹細胞の中から所望の細胞への分化誘導効率が高い細胞を選別することが望まれていた。 (2) Method for selecting induced pluripotent stem cells According to the present invention, induced pluripotent stem cells are induced to differentiate by culturing induced pluripotent stem cells on a structure containing cells and / or components derived from the cells. In addition, induced pluripotent stem cells with high differentiation induction efficiency can be selected. As described above, induced pluripotent stem cells are currently produced by various methods. The created induced pluripotent stem cells are expected to be induced to differentiate into desired cells and applied to regenerative medicine. However, the created artificial pluripotent stem cells usually contain a mixture of cells having high efficiency of inducing differentiation into desired cells and cells having low efficiency of inducing differentiation into desired cells. . It has been desired to select cells with high efficiency of inducing differentiation into desired cells from the prepared induced pluripotent stem cells.
　本発明によれば、細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、所望の細胞への分化誘導効率が高い人工多能性幹細胞を選別することができる。このようにして選別された分化誘導効率が高い人工多能性幹細胞をストックとして保管しておき、再生医療の現場において必要時に分化誘導して使用することができる。 According to the present invention, an induced pluripotent stem cell is induced to differentiate by culturing the induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, and the efficiency of inducing differentiation into a desired cell is improved. Highly induced pluripotent stem cells can be selected. The induced pluripotent stem cells selected in this way and having high differentiation-inducing efficiency can be stored as a stock, and can be differentiated and used when necessary in the field of regenerative medicine.
　また、生体組織又は臓器の切片などの細胞及び／又は細胞に由来する成分を含む構造物は、人工多能性幹細胞の選別のためのキットとして提供することができる。上記キットには、細胞及び／又は細胞に由来する成分を含む構造物以外にも、人工多能性幹細胞を培養するための培地、細胞を含む構造物を設置するためのカバーグラスなどを適宜含めてもよい。 Further, a structure containing cells and / or components derived from cells, such as a section of a living tissue or an organ, can be provided as a kit for selecting induced pluripotent stem cells. In addition to the structure containing cells and / or components derived from the cells, the kit suitably includes a medium for culturing induced pluripotent stem cells, a cover glass for installing structures containing cells, and the like. May be.
　以下の実施例で本発明を詳細に説明するが、本発明はこれらにより限定されるものではない。 The present invention will be described in detail in the following examples, but the present invention is not limited thereto.
（１）凍結切片の作製
　雄性、６週齢のICRマウスから正常な肝臓、脳および脊髄を摘出した。また、1 ml/kgの四塩化炭素をオリーブ油に1:4の割合で混和したものを腹腔内投与することにより人工的に薬剤性肝炎を発症させたICRマウスから、四塩化炭素投与後1、2、3、5日目に肝炎肝臓を摘出した。
　それぞれの臓器をOCTコンパウンドであるTissue-TekR（Sakura Finethechnical）に、包埋、マウントした後、液体窒素に浸漬し凍結切片作成用ブロックを作製した。これらのブロックから、それぞれ厚さ6 μmの凍結切片を作成し、円形のカルチャーカバーグラス（ポリLリジンコートタイプ、松浪ガラス）に載せた。乾燥後、リン酸緩衝液（Phosphate Buffered Saline:以下PBSと略記する、pH 7.4）にて２回洗浄し、OCTコンパウンドを洗い流した後、iPS細胞の培養に用いた。 (1) Preparation of frozen section Normal liver, brain and spinal cord were excised from male, 6-week-old ICR mice. In addition, from ICR mice that artificially developed drug-induced hepatitis by intraperitoneal administration of 1 ml / kg carbon tetrachloride mixed in olive oil at a ratio of 1: 4, after carbon tetrachloride administration, On days 2, 3, and 5, hepatic liver was removed.
Each organ is an OCT compound Tissue-Tek R (Sakura Finethechnical) , embedding, after mounting, to prepare a dipped frozen sectioning block in liquid nitrogen. From these blocks, frozen sections each having a thickness of 6 μm were prepared and placed on a circular culture cover glass (poly L lysine-coated type, Matsunami glass). After drying, the plate was washed twice with a phosphate buffered solution (Phosphate Buffered Saline: hereinafter abbreviated as PBS, pH 7.4) to wash out the OCT compound, and then used for culturing iPS cells.
（２）iPS細胞の培養および分化誘導
　常法に従いiPS細胞を培養後、iPS/ ES細胞剥離液(CTK solution,株式会社リプロセル)を用いてフィーダー細胞を剥離し、PBSにて洗浄後、iPS細胞を回収し、5%ウシ胎児血清（以下FBSと略記する、Biological Industries）を含むダルベッコ改変イーグル培地（以下DMEMと略記する、SIGMA）に懸濁した。35 mm径のディッシュ（ノンコートタイプ、松浪ガラス）に前述の凍結切片を載せた円形カルチャーカバーグラスを設置し、ここにiPS細胞の懸濁液を播種した。播種後1日間静置することで、浮遊しているiPS細胞を凍結切片へ付着させ、その後5%FBSを含むDMEMを2ml加え培養を行った。その後は3日に1度培地交換を行い、計9日間培養した。 (2) iPS cell culture and differentiation induction After culturing iPS cells according to a conventional method, the feeder cells are detached using an iPS / ES cell detachment solution (CTK solution, Reprocell Co., Ltd.), washed with PBS, and then iPS cells. Were collected and suspended in Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM, SIGMA) containing 5% fetal bovine serum (hereinafter abbreviated as FBS, Biological Industries). A circular culture cover glass on which the aforementioned frozen section was placed was placed on a 35 mm diameter dish (non-coated type, Matsunami glass), and a suspension of iPS cells was seeded thereon. By allowing to stand for 1 day after seeding, floating iPS cells were allowed to adhere to the frozen sections, and then 2 ml of DMEM containing 5% FBS was added and cultured. Thereafter, the medium was changed once every 3 days and cultured for a total of 9 days.
　カバーグラス上（対照）、正常肝臓切片上、又は肝炎肝臓切片上で培養したiPS細胞の顕微鏡像（培養２日目と培養８日目）を図１に示す。対照、正常肝臓、肝炎肝臓のいずれも播種したiPS細胞のコロニーが拡がりがり細胞がばらけていくことが認められた。また細胞の形態の変化も認められ、対照では培養２日目と比較すると８日目では拡がったiPS細胞が、矢印で示すように様々な形態を呈し統一性は認められなかった。一方、正常肝臓、肝炎肝臓では拡がった細胞は矢印で示すような比較的大型で多角形の形態を呈する傾向が認められた。
　また、カバーグラス上（対照）又は正常肝臓切片上で培養したiPS細胞の顕微鏡像（培養３日目と９日目）を図２に示し、脳切片上又は脊髄切片上で培養したiPS細胞の顕微鏡像（培養３日目と９日目）を図３に示す。 FIG. 1 shows microscopic images (2nd culture day and 8th culture day) of iPS cells cultured on a cover glass (control), a normal liver section, or a hepatitis liver section. It was observed that the colonies of iPS cells seeded in the control, normal liver, and hepatitis liver spread and the cells spread. In addition, changes in cell morphology were also observed. In the control, iPS cells that had expanded on the 8th day compared to the 2nd day of culture exhibited various forms as indicated by arrows, and no uniformity was observed. On the other hand, in the normal liver and hepatitis liver, the expanded cells showed a relatively large and polygonal shape as indicated by arrows.
In addition, a microscopic image (3rd and 9th culture days) of iPS cells cultured on a cover glass (control) or a normal liver slice is shown in FIG. 2, and the iPS cells cultured on a brain slice or spinal cord slice are shown. A microscopic image (3rd day and 9th day of culture) is shown in FIG.
（３）遺伝子発現の確認
　iPS細胞の肝細胞あるいは神経細胞への分化状態を確認するため、神経細胞および肝細胞の各種分化マーカーのmRNA発現について、半定量的Reverse Transcriptase-Polymerase Chain Reaction法（以下RT-PCR法と略する）を用いて、検討を行った。肝臓、脳、脊髄の凍結切片上で培養を行ったiPS細より、TRIzol reagent（Invitrogen Corp.、Carlsbad、 CA、USA）を用いてtotal RNAを回収した。得られたtotal RNA １μgを室温で100 units/ml デオキシリボヌクレアーゼI（以下DNase Iと略す）で処理した後、1 mlの25 mM EDTAを添加し、65℃、５分間処理し、DNase Iを失活させた。ランダムヘキサマープライマー（Invitrogen）を加え、逆転写酵素SUPERSCRIPT III Preamplification System（Invitrogen）を用いて５０℃、５０分の条件で逆転写反応を行った。さらに３７℃、２０分間リボヌクレアーゼHで処理しcDNA を得た。ヒトの各遺伝子に特異的なプライマーおよびPCR MasterMix Kit（Thermo、Rockford、USA）を用いて、PCRを行い各遺伝子のmRNA発現について検討した。検討した分化マーカー、プライマーおよびPCRの条件を表１に示す。PCR産物は２％アガロースゲルを用いて電気泳動を行い、臭化エチジウムで染色後、UV撮影装置FAS-III（東洋紡績、大阪）を用いて可視化した。 (3) Confirmation of gene expression In order to confirm the differentiation state of iPS cells into hepatocytes or neurons, the semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction method (hereinafter referred to as mRNA expression of various differentiation markers of neurons and hepatocytes) (Abbreviated as RT-PCR method). Total RNA was collected from iPS cells cultured on frozen sections of liver, brain, and spinal cord using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA). 1 μg of the total RNA obtained was treated with 100 units / ml deoxyribonuclease I (hereinafter abbreviated as DNase I) at room temperature, and then 1 ml of 25 mM EDTA was added and treated at 65 ° C. for 5 minutes to lose DNase I. I made it live. A random hexamer primer (Invitrogen) was added, and a reverse transcription reaction was performed at 50 ° C. for 50 minutes using the reverse transcriptase SUPERSCRIPT III Preamplification System (Invitrogen). Further, it was treated with ribonuclease H at 37 ° C. for 20 minutes to obtain cDNA. PCR was performed using primers specific to each human gene and PCR MasterMix Kit (Thermo, Rockford, USA) to examine mRNA expression of each gene. The examined differentiation markers, primers and PCR conditions are shown in Table 1. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and then visualized using a UV imaging device FAS-III (Toyobo, Osaka).
　肝細胞の関連遺伝子（ＡＦＰ、ＡＡＴ、ＡＬＢ）の発現を調べた結果を図４に示す。
AFPの発現は対照群、正常肝臓群、肝炎肝臓群で認められたが、対照群と比較すると肝炎肝臓群で発現が強い傾向がみられ、正常肝臓群ではさらにその発現が増強されていた。AATの発現も対照群、正常肝臓群、肝炎肝臓群で認められたが、対照群と比較すると正常肝臓群で発現が強く認められた。一方、ALBの発現は対照群においては、その発現は認められなかったのに対し、正常肝臓群、肝炎肝臓群では発現が確認され、肝炎肝臓群と比較し正常肝臓群でより強い発現を認めた。この結果から正常肝臓、肝炎肝臓の凍結切片上で培養することによりiPS細胞の固有肝細胞への分化が誘導されていることが確かめられた。
　同様に、神経細胞の関連遺伝子（Nestin、ＭＢＰ、CNPase、及びGFAP）の発現を調べた結果を図５に示す。 The results of examining the expression of hepatocyte-related genes (AFP, AAT, ALB) are shown in FIG.
The expression of AFP was observed in the control group, normal liver group, and hepatitis liver group, but the expression was stronger in the hepatitis liver group than in the control group, and the expression was further enhanced in the normal liver group. AAT expression was also observed in the control group, normal liver group, and hepatitis liver group, but the expression was strongly observed in the normal liver group as compared with the control group. On the other hand, the expression of ALB was not observed in the control group, whereas the expression was confirmed in the normal liver group and hepatitis liver group, and the expression was stronger in the normal liver group than in the hepatitis liver group. It was. From these results, it was confirmed that the differentiation of iPS cells into intrinsic hepatocytes was induced by culturing on frozen sections of normal liver and hepatitis liver.
Similarly, the results of examining the expression of genes related to nerve cells (Nestin, MBP, CNPase, and GFAP) are shown in FIG.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
GAPDH：グリセルアルデヒド-3-リン酸デヒドロゲナーゼ
AFP：α-フェトプロテイン
AAT：α１-アンチトリプシン
ALB：アルブミン
MBP：ミエリン塩基性蛋白
CNPase：2', 3'-環状ヌクレオチド3'-ホスホジエステラーゼ
GFAP：グリア繊維性酸性タンパク質
NES：ネスチン GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
AFP: α-fetoprotein
AAT: α1-antitrypsin
ALB: Albumin
MBP: Myelin basic protein
CNPase: 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase
GFAP: Glial fibrous acidic protein
NES: Nestin
（４）タンパク質発現の確認
　各種凍結切片上で培養したiPS細胞をPBSにて洗浄した後、凍結切片ごと、２% パラホルムアルデヒドにて、室温で６０分間固定を行った。PBSにて洗浄後、１％Triton-Xにて１５分間処理し、１０％正常ウサギ血清または正常ヤギ血清にて室温、１０分間ブロッキングを行った。その後、ウサギ抗ヒトα-フェトプロテイン抗体（Abcam、５００倍希釈）またはヤギ抗ヒトα１-アンチトリプシン抗体（Abcam、３００倍希釈）を４℃、一晩反応させた。PBSにて洗浄後、二次抗体としてFITC-conjugated anti-rabbit IgGまたはFITC-conjugated anti-goat IgGを室温で１時間反応させた。PBSにて洗浄後、核をDAPIにて染色し、蛍光顕微鏡にて観察した（図６及び図８）。図６では、対照群ではAFP陽性細胞はほとんど認められなかったが、正常肝臓群、肝炎肝臓群でAFP陽性細胞が多く認められた。図８では、AFPの場合と同様に対照群と比較し正常肝臓群および肝炎肝臓群でAAT陽性細胞が多く認められた。α-フェトプロテインおよびα１-アンチトリプシンを発現しているiPS細胞の数を計測、有核細胞に占める割合を算出することにより（図７及び図９）、分化誘導効率を検討した。図７に示す通り、対照群に比較して正常肝臓群及び肝炎肝臓１日目の群で、AFP陽性細胞の割合が有意に多く認められた。また、図９に示す通り、対照群に比較して、正常肝臓群、肝炎肝臓１，２，５日目の群でAAT陽性細胞の割合が有意に多く認められた。 (4) Confirmation of protein expression After washing iPS cells cultured on various frozen sections with PBS, the frozen sections were fixed with 2% paraformaldehyde at room temperature for 60 minutes. After washing with PBS, the cells were treated with 1% Triton-X for 15 minutes, and blocked with 10% normal rabbit serum or normal goat serum at room temperature for 10 minutes. Thereafter, a rabbit anti-human α-fetoprotein antibody (Abcam, diluted 500 times) or a goat anti-human α1-antitrypsin antibody (Abcam, diluted 300 times) was reacted at 4 ° C. overnight. After washing with PBS, FITC-conjugated anti-rabbit IgG or FITC-conjugated anti-goat IgG was reacted as a secondary antibody for 1 hour at room temperature. After washing with PBS, nuclei were stained with DAPI and observed with a fluorescence microscope (FIGS. 6 and 8). In FIG. 6, almost no AFP positive cells were observed in the control group, but many AFP positive cells were observed in the normal liver group and the hepatitis liver group. In FIG. 8, as in the case of AFP, more AAT positive cells were observed in the normal liver group and the hepatitis liver group than in the control group. The differentiation induction efficiency was examined by measuring the number of iPS cells expressing α-fetoprotein and α1-antitrypsin and calculating the proportion of nucleated cells (FIGS. 7 and 9). As shown in FIG. 7, the ratio of AFP positive cells was significantly higher in the normal liver group and the group on the first day of hepatitis liver than in the control group. In addition, as shown in FIG. 9, the proportion of AAT positive cells was significantly higher in the normal liver group and the hepatitis liver 1, 2 and 5 day groups than in the control group.
　また、一次抗体として、抗ヒトGFAP抗体又は抗CNPase抗体を使用して、タンパク質発現を蛍光顕微鏡にて観察した。各種凍結切片上で培養したiPS細胞を、2％パラホルムアルデヒドにて１時間固定し、 PBSで2回洗浄した後、0.1% BSA含有PBSにて希釈した1% TRITON X-100 (ICN Biomedical)にて15分処理した。PBSで2回洗浄後、10%ヤギ正常血清にて1時間ブロッキングした。PBSで2回洗浄後、1% BSA含有PBSにて希釈した一次抗体にて４℃で一晩反応させた。一次抗体としては、Rabbit anti-Glial Fibrillary Acidic Protein antibody (SIGMA、８０倍希釈）又はRabbit anti-CNPase antibody (Abcam、１００倍希釈）を使用した。PBSで3回洗浄後、1% BSA含有PBSにて希釈した二次抗体にて１時間反応させた。二次抗体としては、GFAPについては、Goat anti-rabbit IgG FITC (Wako、４０倍希釈)を使用し、CNPaseについては、Goat anti-rabbit IgG Alexa（Invitergen、１００倍希釈）を使用した。 PBSで2回洗浄後、DAPIにて30分染色した。PBSで2回洗浄し、VECTASHIELD にて封入し、蛍光顕微鏡にて観察した。 In addition, protein expression was observed with a fluorescence microscope using an anti-human GFAP antibody or an anti-CNPase antibody as a primary antibody. IPS cells cultured on various frozen sections were fixed with 2% paraformaldehyde for 1 hour, washed twice with PBS, and then diluted with 0.1% BSA-containing PBS to 1% TRITON X-100 (ICN Biomedical). For 15 minutes. After washing twice with PBS, blocking was performed with 10% normal goat serum for 1 hour. After washing twice with PBS, the mixture was reacted overnight at 4 ° C. with a primary antibody diluted with PBS containing 1% BSA. As the primary antibody, Rabbit anti-Glial Bibrillary Acidic Protein antibody (SIGMA, 80-fold dilution) or Rabbit anti-CNPase antibody (Abcam, 100-fold dilution) was used. After washing 3 times with PBS, the mixture was reacted for 1 hour with a secondary antibody diluted with PBS containing 1% BSA. As a secondary antibody, Goat anti-rabbit IgG FITC (Wako, diluted 40-fold) was used for GFAP, and Goat anti-rabbit IgG Alexa (Invitergen, diluted 100-fold) was used for CNPase. After washing twice with PBS, stained with DAPI for 30 minutes. The plate was washed twice with PBS, sealed with VECTASHIELD, and observed with a fluorescence microscope.
　一次抗体として、抗ヒトGFAP抗体を使用してタンパク質発現を蛍光顕微鏡にて観察した結果を図１０に示す。GFAPを発現しているiPS細胞の数を計測、有核細胞に占める割合を算出することにより（図１１）、分化誘導効率を検討した。
　一次抗体として、抗CNPase抗体を使用してタンパク質発現を蛍光顕微鏡にて観察した結果を図１２及び図１３に示す。CNPaseを発現しているiPS細胞の数を計測、有核細胞に占める割合を算出することにより（図１４）、分化誘導効率を検討した。 FIG. 10 shows the results of observation of protein expression with a fluorescence microscope using an anti-human GFAP antibody as a primary antibody. The number of iPS cells expressing GFAP was counted, and the proportion of nucleated cells was calculated (FIG. 11), and the differentiation induction efficiency was examined.
The results of observation of protein expression with a fluorescence microscope using an anti-CNPase antibody as the primary antibody are shown in FIGS. The differentiation induction efficiency was examined by measuring the number of iPS cells expressing CNPase and calculating the proportion of nucleated cells (FIG. 14).
　カバーグラス上（対照）、正常肝臓切片上、又は肝炎肝臓切片上でiPS細胞を培養した場合について、細胞形態及び遺伝子（ＡＦＰ、ＡＡＴ、ＡＬＢ）発現の結果を表２に示し、タンパク質（ＡＦＰ、ＡＡＴ）発現の結果を表３に示す。また、ＧＦＡＰとＣＮＰａｓｅの遺伝子及びタンパク質発現の結果を表４に示す。 The results of cell morphology and gene (AFP, AAT, ALB) expression for iPS cells cultured on cover glass (control), normal liver sections, or hepatitis liver sections are shown in Table 2, and proteins (AFP, The results of AAT) expression are shown in Table 3. In addition, Table 4 shows the results of GFAP and CNPase gene and protein expression.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　上記した結果から、本発明の方法により、ｉＰＳ細胞から肝細胞及び神経細胞への分化誘導が可能であることが示された。 From the above results, it was shown that differentiation of iPS cells into hepatocytes and neurons can be induced by the method of the present invention.

Claims (16)

  1. 細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む人工多能性幹細胞の分化誘導方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の分化誘導方法。 A method for inducing differentiation of an induced pluripotent stem cell comprising culturing an induced pluripotent stem cell on a structure containing a cell and / or a component derived from the cell, wherein the cell is differentiated from the cell in the structure And the differentiation induction method.
  2. 細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、請求項１に記載の方法。 The method of Claim 1 that the structure containing the component derived from a cell and / or a cell is a sheet-like structure.
  3. 細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the cell and / or the structure containing a cell-derived component is a biological tissue, a section of an organ, or a culture substrate coated with a cell-derived component.
  4. 細胞が、肝臓、脳又は脊髄である、請求項１から３の何れか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the cells are liver, brain or spinal cord.
  5. 細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することを含む、分化誘導された細胞の製造方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の製造方法。 A method for producing differentiation-induced cells, comprising culturing induced pluripotent stem cells on a structure containing cells and / or components derived from the cells, wherein the cells in the structure and the differentiation-induced cells And the above-mentioned production method.
  6. 細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、請求項５に記載の方法。 The method of Claim 5 that the structure containing the component derived from a cell and / or a cell is a sheet-like structure.
  7. 細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項６又は７に記載の方法。 The method according to claim 6 or 7, wherein the structure containing a cell and / or a component derived from a cell is a culture substrate coated with a tissue derived from a living tissue, an organ section or a cell.
  8. 細胞が、肝臓、脳又は脊髄である、請求項５から７の何れか１項に記載の方法。 The method according to any one of claims 5 to 7, wherein the cell is liver, brain or spinal cord.
  9. 細胞及び／又は細胞に由来する成分を含む構造物上で人工多能性幹細胞を培養することによって人工多能性幹細胞を分化誘導し、分化誘導効率が高い人工多能性幹細胞を選別することを含む人工多能性幹細胞の選別方法であって、構造物中の細胞と分化誘導された細胞とが同じ細胞型である前記の選別方法。 Inducing differentiation of induced pluripotent stem cells by culturing induced pluripotent stem cells on a structure containing cells and / or cell-derived components, and selecting induced pluripotent stem cells with high differentiation induction efficiency A method for sorting induced pluripotent stem cells, wherein the cells in the structure and the cells induced to differentiate are of the same cell type.
  10. 細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、請求項９に記載の方法。 The method of Claim 9 that the structure containing the component derived from a cell and / or a cell is a sheet-like structure.
  11. 細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項９又は１０に記載の方法。 The method according to claim 9 or 10, wherein the cell and / or the structure containing a cell-derived component is a biological tissue, a section of an organ, or a culture substrate coated with a cell-derived component.
  12. 細胞が、肝臓、脳又は脊髄である、請求項９から１１の何れか１項に記載の方法。 The method according to any one of claims 9 to 11, wherein the cell is liver, brain or spinal cord.
  13. 細胞及び／又は細胞に由来する成分を含む構造物を少なくとも含む、人工多能性幹細胞の選別のためのキット。 A kit for selecting induced pluripotent stem cells, comprising at least a structure containing cells and / or components derived from cells.
  14. 細胞及び／又は細胞に由来する成分を含む構造物がシート状構造物である、請求項１３に記載のキット。 The kit according to claim 13, wherein the structure containing cells and / or components derived from cells is a sheet-like structure.
  15. 細胞及び／又は細胞に由来する成分を含む構造物が生体組織、臓器の切片または細胞に由来する成分をコーティングした培養基材である、請求項１３又は１４に記載のキット。 15. The kit according to claim 13 or 14, wherein the structure containing cells and / or components derived from cells is a culture substrate coated with biological tissue, organ sections or cells-derived components.
  16. 細胞が、肝臓、脳又は脊髄である、請求項１３から１５の何れか１項に記載のキット。 The kit according to any one of claims 13 to 15, wherein the cells are liver, brain or spinal cord.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005520516A (en) 2002-03-15 2005-07-14 モナシュ・ユニヴァーシティ Methods for inducing differentiation of stem cells into specific cell lineages
WO2007069666A1 (en) 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor
US20080233610A1 (en) 2007-03-23 2008-09-25 Thomson James A Somatic cell reprogramming
WO2008118220A2 (en) 2006-11-28 2008-10-02 Veritainer Corporation Radiation detection unit for mounting a radiation sensor to a container crane
WO2008124133A1 (en) 2007-04-07 2008-10-16 Whitehead Institute For Biomedical Research Reprogramming of somatic cells
WO2008151058A2 (en) 2007-05-30 2008-12-11 The General Hospital Corporation Methods of generating pluripotent cells from somatic cells
JP2008307007A (en) 2007-06-15 2008-12-25 Bayer Schering Pharma Ag Human pluripotent stem cell induced from human tissue-originated undifferentiated stem cell after birth
US20090047263A1 (en) 2005-12-13 2009-02-19 Kyoto University Nuclear reprogramming factor and induced pluripotent stem cells
WO2009057831A1 (en) 2007-10-31 2009-05-07 Kyoto University Nuclear reprogramming method

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102712900B (en) * 2009-10-06 2015-07-01 首尔大学校产学协力团 Method for differentiation into retinal cells from stem cells
US20130315962A1 (en) * 2010-07-06 2013-11-28 Nanologica Ab Method for stem cell differentiation in vivo by delivery of morphogenes with mesoporous silica and corresponding pharmceutical active ingredients
WO2012043814A1 (en) * 2010-09-30 2012-04-05 国立大学法人東京大学 Method for differentiating human-derived pluripotent stem cells
US11326150B2 (en) * 2011-09-27 2022-05-10 Public University Corporation Yokohama City University Method for producing tissue and organ

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005520516A (en) 2002-03-15 2005-07-14 モナシュ・ユニヴァーシティ Methods for inducing differentiation of stem cells into specific cell lineages
WO2007069666A1 (en) 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor
JP2008283972A (en) 2005-12-13 2008-11-27 Kyoto Univ Method for producing induced pluripotent stem cell
US20090047263A1 (en) 2005-12-13 2009-02-19 Kyoto University Nuclear reprogramming factor and induced pluripotent stem cells
WO2008118220A2 (en) 2006-11-28 2008-10-02 Veritainer Corporation Radiation detection unit for mounting a radiation sensor to a container crane
US20080233610A1 (en) 2007-03-23 2008-09-25 Thomson James A Somatic cell reprogramming
WO2008124133A1 (en) 2007-04-07 2008-10-16 Whitehead Institute For Biomedical Research Reprogramming of somatic cells
WO2008151058A2 (en) 2007-05-30 2008-12-11 The General Hospital Corporation Methods of generating pluripotent cells from somatic cells
JP2008307007A (en) 2007-06-15 2008-12-25 Bayer Schering Pharma Ag Human pluripotent stem cell induced from human tissue-originated undifferentiated stem cell after birth
WO2009006997A1 (en) 2007-06-15 2009-01-15 Izumi Bio, Inc. Human pluripotent stem cells and their medical use
WO2009007852A2 (en) 2007-06-15 2009-01-15 Izumi Bio, Inc Multipotent/pluripotent cells and methods
WO2009057831A1 (en) 2007-10-31 2009-05-07 Kyoto University Nuclear reprogramming method

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
EDITED BY SHIN'YA YAMANAKA ET AL., ES SAIBO KARA NO BUNKA, 20 October 2012 (2012-10-20), SAISEI IRYO SOSHO 1 KANSAIBO, pages 21 - 29, XP008183198 *
MASANORI TAKENAGA: "Suizo tono Kyobaiyo ni yoru Mouse ES Saibo no Naihaiyo eno Bunka Yudo", JOURNAL OF JAPAN SURGICAL SOCIETY, vol. 106, 5 April 2005 (2005-04-05), pages 284, XP008183147 *
NAKAGAWA M; YAMANAKA S. ET AL., NATURE BIOTECHNOLOGY, vol. 26, 2008, pages 101 - 106
NATHANIEL S. ET AL.: "Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration", PLOS ONE, vol. 3, no. 6, 25 June 2008 (2008-06-25), pages E2498, XP055084312 *
PARK M; DALEY GQ ET AL., NATURE, vol. 451, 2007, pages 141 - 146
TAKAHASHI K; YAMANAKA S. ET AL., CELL, vol. 131, 2007, pages 861 - 872
TAKAHASHI K; YAMANAKA S., CELL, vol. 126, 2006, pages 663 - 676
YU J.; THOMSON JA ET AL., SCIENCE, vol. 318, 2007, pages 1917 - 1920

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